Mechanisms of Klebsiella pneumoniae gastrointestinal colonization

摘要 由耐药病原体传播引起的医院获得性感染(HAI)影响数百人 全球数以百万计的患者。肺炎克雷伯氏菌(Kpn)是一种革兰氏阴性细菌，因 引起HAI，其中许多感染很难治疗，因为KPN已经变得对多种药物具有耐药性。 流行病学研究表明，KPN的胃肠道(GI)定植是一个主要的宿主，通过 哪种KPN可以在被定植的宿主中引起疾病表现，或者在宿主之间传播。此站点 作为KPN GI定殖化的易处理模型，KPN定殖率并不是先前研究的重点，并且 主机到主机的传输不存在。我们最近开发了一种小鼠模型，可以进行这项研究 KPN粘膜(口咽和胃肠道)的定植、在粪便中的脱落和通过粪便-口腔的传播 路线。使用口服接种和排泄物的途径作为胃肠道定植的标志，我们展示了 KPN可无症状地定植于免疫活性小鼠的胃肠道并改变宿主胃肠道 微生物区系。我们假设特定的KPN基因有助于其在胃肠道的定植，以及这些基因的产物 可作为防止建立大军殖民的新目标。最近，我们使用了 我们的小鼠模型筛选KPN随机转座子突变体文库(In-seq)以确定完整的 来自单一分离物的“GI定植”基因。元基因组测序分析进一步鉴定出细菌 胃肠道中受KPN影响的物种和代谢途径。在这里，我们将重点关注两组路径 通过In-Seq认证，其产品允许KPN克服居民提供的殖民阻力 肠道微生物区系。因此，在目标1中，我们将重点研究KPN的乙醇胺利用途径基因(EUT)，该基因 允许它利用乙醇胺(EA)，乙醇胺是肠道中细胞膜和饮食的副产品，可以作为 替代营养源。与许多其他只含有一个eut操纵子的肠道病原体不同，KPN有两个操纵子 基因上截然不同的EUT操纵子。我们将确定每个EUT基因座在EA代谢中的作用，并确定 EA代谢为KPN提供适应性优势的潜在分子机制 微生物组的成员。目标2将采取一种不同的方法，专注于接触依赖型杀戮 KPN(6型分泌系统[T6SS])克服由KPN提供的定植抗性的机制 常驻微生物区系。我们将重点研究调控表达的独特调控机制 KPN T6SS在胃肠道中的分布，并为其提供相对于居民肠道的选择性和竞争优势 微生物区系。这些研究的结果将使我们对分子有一个基本的了解 由传入病原体建立胃肠道定植的机制。这些研究还将 为制定降低KPN疾病负担的潜在战略奠定基础。

Abstract Hospital-acquired infections (HAI) resulting from the transmission of drug-resistant pathogens affect hundreds of millions of patients worldwide. Klebsiella pneumoniae (Kpn), a gram-negative bacterium, is notorious for causing HAI, with many of these infections difficult to treat as Kpn has become multi-drug resistant. Epidemiological studies suggest that gastrointestinal (GI) colonization of Kpn is a major reservoir through which Kpn can cause disease manifestations either in the colonized host or transmit from host to host. This site of Kpn colonization has not been the focus of previous studies as a tractable model of Kpn GI colonization, and host-to-host transmission did not exist. We have recently developed a murine model that allows for the study of Kpn mucosal (oropharynx and GI) colonization, shedding within feces, and transmission through the fecal-oral route. Using an oral route of inoculation and fecal shedding as a marker for GI colonization, we show that Kpn can asymptomatically colonize the GI tract of immunocompetent mice and modifies the host GI microbiota. We premise that specific Kpn genes contribute to its GI colonization, and the products of these genes could serve as novel targets for the prevention of the establishment of GI colonization. More recently, we used our murine model to screen a library of Kpn random transposon mutants (In-seq) to identify the complete set of “GI colonization” genes from a single isolate. A metagenomics sequencing analysis further identified bacterial species and the metabolic pathways affected by Kpn in the GI tract. Herein, we will focus on two sets of pathways identified through In-seq whose products allow Kpn to overcome colonization resistance provided by the resident gut microbiota. Thus, in Aim#1, we will focus on the ethanolamine utilization pathway genes (eut) of Kpn that allow it to utilize ethanolamine (EA), a byproduct of cellular membranes and diet in the gut that can serve as an alternative nutrient source. Unlike many other enteric pathogens that contain a single eut operon, Kpn has two genetically distinct eut operons. We will identify the role of each eut locus in EA metabolism and determine the underlying molecular mechanism through which EA metabolism provides Kpn with a fitness advantage against members of the microbiome. Aim#2 will take a different approach by focusing on the contact-dependent killing machinery of the Kpn (Type 6 secretion system [T6SS]) in overcoming colonization resistance provided by the resident microbiota. We will focus on the unique regulatory mechanism that modulates the expression of Kpn T6SS in the GI tract and provide it with a selective and competitive advantage against the resident gut microbiota. Results from these studies would provide us with a fundamental understanding of the molecular mechanisms involved in the establishment of GI colonization by an incoming pathogen. These studies will also lay the groundwork for developing potential strategies to reduce the Kpn disease burden.